付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(理学) / 甲第24471号 / 理博第4970号 / 新制||理||1709(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 伊谷 原一, 教授 平田 聡 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

In early 2020 the World Health Organization (WHO) declared a COVID-19 pandemic outbreak. As of March 2023, the Kingdom of Saudi Arabia has reported over 829 thousand cases and over 9 thousand deaths. Due to the public health emergency, the wild-type SARS-CoV-2 genome was fully sequenced early on the pandemic course. Afterward, different viral variants emerged around the world. The variant prevalence fluctuates over time, with some strains presenting increased transmissibility, changes in disease severity, and decreased efficacy of containment measures. In this respect, genomic surveillance and timely data sharing to public repositories represent a tool to track and detect emerging changes that require addressing with public health measurements. However, the Kingdom's genome contribution to the GISAID database represents merely 0.27% of the reported cases. To address this gap, we incorporated whole genome sequencing and the available metadata, to track the circulating viral lineages and identify the variants of concern (VOC) and variants of interest (VOI) over 14 months, from December 2021 to January 2023. We sequenced 581 genomes using the MinION MK1C platform of Oxford Nanopore Technologies (ONT) and assigned the viral lineages, the data primarily derived from COVID-19-positive patients from the city of Jeddah. The VOC Omicron was the principal circulating variant in the country (~99%), which aligns with the global trend. Our report included the sublineages BA.2.75, BQ.1, CH.1.1, XBB, and XBB.1.5, which are under WHO monitoring. We also reported the VOC Delta and the recombinant strains XF, XZ, and XPB. Finally, to validate the use of ONT for genomic surveillance, we compared the data quality of 93 samples sequenced in both ONT MinION and Illumina NaovaSeq platforms. There was 90% correspondence in the Nextstrain clade assignment (84/93) and 78% correspondence in the Pangolin sublineage assignment (73/93). MinION offered a shorter turnaround time, while Illumina produced a consistently higher breadth of genome coverage. In conclusion, the continued viral evolution patterns are reflected in the Kingdom's variant prevalence, where only Omicron circulated by January 2023. Likewise, current Variants Under Monitoring (VUM) were already identified in KSA, highlighting the need for increased genomic surveillance.

Yes / Aeromonas hydrophila has emerged worldwide as a human pathogen. Here, we report the draft whole-genome sequence of a freshwater isolate from Malaysia, A. hydrophila strain M062, and its N-acylhomoserine lactone genes are also reported here. / University of Malaya via High-Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01, no. A-000001- 50001), and aUM-MOHEHIR grant (UM C/625/1/HIR/MOHE/CHAN/ 14/1, no. H-50001-A000027)

Yes / Trypanosomes cause a variety of diseases in man and domestic animals in Africa, Latin America, and Asia. In the Trypanozoon subgenus, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis, whereas Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum are responsible for nagana, surra, and dourine in domestic animals, respectively. The genetic relationships between T. evansi and T. equiperdum and other Trypanozoon species remain unclear because the majority of phylogenetic analyses has been based on only a few genes. In this study, we have conducted a phylogenetic analysis based on genome-wide SNP analysis comprising 56 genomes from the Trypanozoon subgenus. Our data reveal that T. equiperdum has emerged at least once in Eastern Africa and T. evansi at two independent occasions in Western Africa. The genomes within the T. equiperdum and T. evansi monophyletic clusters show extremely little variation, probably due to the clonal spread linked to the independence from tsetse flies for their transmission. / Funding was received from the Research Foundation Flanders (FWO, grants 1501413N and 1101614N) and the European DG Health and Food Safety (SANTE). We thank the Center of Medical Genetics at the University of Antwerp for hosting the NGS facility.

Yes / Background: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. Methods: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005–2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. Findings: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. Interpretation: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting. / ERC grant [INTERRUPTB; no. 311725] to BdJ, FG and CJM; an ERC grant to TS [PhyPD; no. 335529]; an FWO PhD fellowship to PM [grant number 1141217N]; the Leibniz Science Campus EvolLUNG for MM and SN; the German Centre for Infection Research (DZIF) for TAK, MM, CU, PB and SN; a SNF SystemsX grant (TBX) to JP and TS and a Marie Heim-Vögtlin fellowship granted to DK by the Swiss National Science Foundation. The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation - Flanders (FWO) and the Flemish Government – department EWI.

Yes / We investigated whether companion drug resistance was associated with adverse outcome of the shorter MDR-TB regimen in Bangladesh, after adjusting for fluoroquinolone resistance. MDR/RR-TB patients registered for treatment with a standardized gatifloxacin-based shorter MDR-TB regimen were selected for the study. Drug resistance was determined using the proportion method, gatifloxacin and isoniazid minimum inhibitory concentration testing for selected isolates, and whole genome sequencing. Low-level and high-level fluoroquinolone resistance were the most important predictors of adverse outcomes, with pyrazinamide resistance having a significant yet lower impact. In patients with fluoroquinolone-/second-line injectable-susceptible TB, non-eligibility to the shorter MDR-TB regimen (initial resistance to either pyrazinamide, ethionamide, or ethambutol) was not associated with adverse outcome (aOR 1.01; 95%CI 0.4-2.8). Kanamycin resistance was uncommon (1.3%). Increasing levels of resistance to isoniazid predicted treatment failure, also in a subgroup of patients with high-level fluoroquinolone-resistant TB. Our results suggest that resistance to companion drugs of the shorter MDR-TB regimen, except kanamycin resistance, is of no clinical importance as long as fluoroquinolone susceptibility is preserved. Hence, contrary to current WHO guidelines, exclusions to the standard regimen are justified only in the case of fluoroquinolone, and possibly kanamycin resistance. / Damien Foundation Belgium for its financial and logistic support to run the project including its research activities. European Research Council (Starting Grant INTERRUPTB 311725).

yes / BACKGROUND: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis allows rapid, accurate inferences about the sources, location and timing of transmission. However, in an era of heightened concern for personal privacy and science distrust, such inferences could result in unintended harm and undermine the public´s trust. METHODS: We held interdisciplinary stakeholder discussions and performed ethical analyses of real-world illustrative cases to identify principles that optimise benefit and mitigate harm of M. tuberculosis WGS-driven TB source investigations.RESULTS: The speed and precision with which real-time WGS can be used to associate M. tuberculosis strains with sensitive information has raised important concerns. While detailed understanding of transmission events could mitigate harm to vulnerable patients and communities when otherwise unfairly blamed for TB outbreaks, the precision of WGS can also identify transmission events resulting in social blame, fear, discrimination, individual or location stigma, and the use of defaming language by the public, politicians and scientists. Public health programmes should balance the need to safeguard privacy with public health goals, transparency and individual rights, including the right to know who infects whom or where.CONCLUSIONS: Ethical challenges raised by real-time WGS-driven TB source investigation requires public health authorities to move beyond their current legal mandate and embrace transparency, privacy and community engagement.

No description available.

Avian pathogenic Escherichia. coli (APEC) and Campylobacter are pathogenic threats to poultry and human health, respectively. In this study, the prevalence of these pathogens in Mississippi broilers and their antimicrobial resistance (AMR) properties were investigated, and a multidrug-resistant APEC strain (APEC-O2-MS1170) was further explored by whole-genome sequencing (WGS). The efficacy of in ovo injection of Lactobacillus in reducing the APEC in broilers was evaluated. Results revealed a high prevalence of APEC and Campylobacter in broilers and broiler products. A lot of isolates were resistant to antibiotics of different sorts. Moreover, the in ovo administration of Lactobacillus did not reduce the incidence of APEC. The WGS of APEC-O2-MS1170 revealed its detailed AMR and virulence properties and alerted a potential zoonotic risk. In conclusion, the Lactobacillus did not reduce the incidence of APEC in broilers, and the prevalence and AMR of APEC and Campylobacter are still challenges faced by the poultry industry.

Infectious diseases cause substantial morbidity and mortality, exacerbated by increasing antibiotic resistance. In critically ill patients, recent studies indicate a substantial variability in β-lactam antibiotic levels when standardized dosing is applied. New methods for characterizing nosocomial outbreaks of bacterial infections are needed to limit transmission. The goals of this thesis were to investigate new strategies towards individualized treatment and control of bacterial infections.  In Paper I we confirmed high variability in β-lactam antibiotic levels among intensive care unit (ICU) patients from southeastern Sweden, where 45 % failed to reach treatment targets (100 % fT>MIC). Augmented renal clearance and establishing the minimum inhibitory concentration of the bacteria were important for evaluating the risk of not attaining adequate drug levels. In Paper II a rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 11 commonly used antibiotics was developed and tested in clinical samples. Performance goals (CV<15%) were reached. A microbiological method for quantification of β-lactam antibiotics in serum was developed in Paper III. The method could be important for hospitals without access to an LC-MS method. Paper IV and Paper V investigated ligation-mediated qPCR with high resolution melt analysis (LMqPCR HRMA), for transmission investigation of extended spectrum β-lactamase (ESBL)-producing E. coli and other common bacterial pathogens. Results comparable to the reference method (PFGE) could be achieved within one day in a closed system and confirmed a nosocomial outbreak in Kalmar County. In Paper VI whole genome sequencing followed by bioinformatic analysis resolved transmission links within a nosocomial outbreak due to improved discriminatory power compared to LMqPCR HRMA. The high proportion of ICU patients with insufficient β-lactam drug levels emphasizes the need for individualized treatment by therapeutic drug monitoring (TDM). TDM is enabled by a highly sensitive method, such as UPLC-MS/MS, but if unavailable, also by a microbial method. Molecular typing methods used for transmission investigation can detect nosocomial outbreaks. LMqPCR HRMA can be used for screening purposes. For enhanced resolution, whole genome sequencing should be used, but always together with a rigorous epidemiological investigation.